RFA-03-CA-101. Challenge Area: Biomarker Discovery and Validation. Challenge Topic: Fingerprints for the Early Detection and Treatment of Cancer. Title: "Early Functional Genetic and Epigenetic Changes in Human Lung Carcinogenesis." Lung carcinogenesis is initiated with the first cigarette, but the sequence and pathways of gene dysregulation events in smokers leading to early disease remain obscure. Our group has developed several assays at the epigenome-wide level and locus-specific level that allow unique insights into gene dysregulation. We hypothesize that a transcriptome and epigenome-wide search among histologically divergent lung-epithelial phenotypes will reveal candidate lung carcinogenesis marks, some of which will be confirmed by high resolution assays as key regulatory loci underlying lung carcinogenesis. In an initial and then confirmatory set of lung cancer case subjects, we will generate initial discovery transcriptome, and epigenome-wide marks of possible phenotypic significance that will then be verified and refined with high density epigenetic reference techniques. This will allow identification of relevant, phenotype defining epigenome marks at the DNA methylation, and microRNA levels. For initial gene expression changes, we will assay (1) mRNA levels by expression microarrays, that are then verified by laboratory-developed RNA specific mRNA-PCR. For assaying the epigenome, we will employ: (2) Methylome-wide HpaII tiny fragment Enrichment Ligation-mediated PCR (HELP) assay, with phenotype-differentiating loci verified by deeper sequencing using mass spectroscopy-based sequencing (MassArray(r)) and re-verified at key loci using the laboratory-developed tagged bisulfite genomic sequencing (tBGS);(3) Micronome-wide assays of microRNA expression by parallel sequencing, verified by both microRNA-qPCR and microRNA pull-down as developed in our laboratory. The key concordant marks for both expression and epigenetic features will be reconfirmed in fluorescent bronchoscopy-guided brush-exfoliated bronchial cells of normal, dysplastic, and malignant epithelium from a subset of donors. The project will therefore reveal comprehensive and unique patterns of gene dysregulation inherent to different steps of lung carcinogenesis in those individuals who prove to have that propensity (lung cancer cases). These studies are a prelude to the development of risk and early diagnostic airway biomarkers, in future prospective studies funded by other mechanisms. PUBLIC HEALTH RELEVANCE: This project is aimed at discovering gene expression and epigenetic gene regulation features that are important for the transition of human lung epithelium from normal to precancerous to cancerous. In parallel studies, we are actively developing non-invasive approaches to measuring these same epigenetic lung carcinogenesis biomarkers in humans for direct clinical studies. The goal is to use the epigenetic features for diagnostic, preventive, and therapeutic purposes.